Effects of Exogenous Interleukin-7 on Human Thymus Function

IMMUNOBIOLOGY

Effects of exogenous interleukin-7 on human thymus function

Yukari Okamoto, Daniel C. Douek, Richard D. McFarland, and Richard A. Koup

Immune reconstitution is a critical compo- (14 692 copies/10 000 cells) was present ments using NOD/LtSz-scid mice given nent of recovery after treatment of human in the CD1a؉CD3؊CD4؉CD8؉ stage in na- transplants of human fetal thymus and immunodeﬁciency virus (HIV) infection, tive thymus, suggesting that TREC gen- liver suggested that IL-7 can also directly cancer chemotherapy, and hematopoietic eration occurred following the cellular enhance TREC generation. Our results stem cell transplantation. The ability to division in this subpopulation. In a thymic provide compelling evidence that IL-7 has enhance T-cell production would beneﬁt organ culture system, exogenous IL-7 a direct effect on increasing TCR-␣␤ rear- such treatment. We examined the effects increased the TREC frequency in fetal as rangement and indicate the potential use of exogenous interleukin-7 (IL-7) on apo- well as infant thymus, indicating in- of IL-7 for enhancing de novo naı¨ve T-cell ptosis, proliferation, and the generation creased T-cell receptor (TCR) rearrange- generation in immunocompromised pa- of T-cell receptor rearrangement excision ment. Although this increase could be tients. (Blood. 2002;99:2851-2858) circles (TRECs) in human thymus. Quanti- due to the effect of IL-7 to increase thymo- tative polymerase chain reaction demon- cyte proliferation and decrease apoptosis strated that the highest level of TRECs of immature CD3؊ cells, the in vivo experi- © 2002 by The American Society of Hematology Introduction

It has been reported that T-cell numbers are maintained in adults recombination event, which is common to approximately 70% of predominantly through the expansion of postthymic, memory thymocytes destined to become mature TCR␣␤ T cells. TRECs are T cells, whereas in infants, T cells are predominantly maintained stable and do not replicate with cellular proliferation,2,13; therefore, through the production of new naı¨ve T cells by the thymus.1 the TREC content in a peripheral cell population is proportional to However, others and we have recently demonstrated that the adult the frequency of recent thymic emigrants (RTEs). This frequency, thymus is still capable of thymopoiesis and can contribute to T-cell however, is affected not only by changes in thymic output but also reconstitution in adults.2,3 Several methods have been used to by the proliferative history of the cells. Because RTEs have TRECs measure thymopoietic capacity. Thymic size as measured by at levels that would not have been affected by peripheral expansion radiographic imaging1 and volumetric computed tomography mea- and cellular replication, quantification of TREC levels in peripheral surements4,5 have been correlated with numbers of CD4ϩCD45RAϩ blood mononuclear cells (PBMCs) represents a sensitive measure- naı¨ve T cells, and the number of phenotypically naı¨ve T cells after ment of thymopoietic capacity. transplantation has been shown to correlate with antigen-specific Although thymic function declines with age, substantial output function.6 However, there are concerns about limitations of estimat- is maintained into late adulthood.2,3 Furthermore, the adult thymus ing thymic function based on naı¨ve T-cell phenotype alone. T cells can contribute to immune reconstitution in individuals following expressing a naı¨ve phenotype are not necessarily accurate surrogate antiretroviral therapy,2,14,15 and following myeloablative chemo- markers of thymic function. Following thymic emigration, therapy and autologous hematopoietic stem cell transplantation.16 CD45RAϩ naı¨ve T cells can have a long quiescent life span,7 may T cells generated de novo from thymopoiesis have a broad TCR proliferate in an antigen-independent manner,8 or may rapidly repertoire and are theoretically more capable of responding to convert to CD45ROϩ memory/effector phenotype T cells.9 Further- neoantigens effectively.16,17 In contrast, peripheral expansion of more, naı¨ve T-cell markers may be acquired by memory T cells existing T-cell pools may lead to limited T-cell repertoires and (especially CD8ϩ T cells),9,10 further compounding the difficulty in antigen responsiveness.1,17-22 Therefore, in patients with human accurately enumerating naı¨ve T cells.11,12 immunodeficiency virus (HIV) infection or in those who have To measure thymic function more directly in humans, we received chemotherapy, the ability of the thymus to generate naı¨ve recently described an assay that quantifies an episomal DNA T cells with a broad TCR repertoire should allow for recovery of T by-product of the T-cell receptor (TCR) rearrangement process.2 cell–mediated immunity that is qualitatively better than if the These TCR rearrangement excision circles (TRECs) contain the recovery were only through expansion of pre-existing naı¨ve and signal joint sequences from the TCRAD locus ␦Rec to ␺J␣ memory T cells.

From the Vaccine Research Center, National Institute of Allergy and Infectious 02680-28-RGV (to D.C.D.). The opinions expressed are those of the author Diseases and Department of Experimental Transplantation and Immunology, (R.D.M.) and not necessarily those of the US Food and Drug Administration. Medicine Branch, National Cancer Institute, National Institutes of Health, Reprints: Richard A. Koup, Immunology Laboratory, National Institutes of Bethesda, MD; and the Center for Biologics Evaluation and Research, Food Health, Vaccine Research Center, Rm 3502, 40 Convent Dr, Bethesda, MD and Drug Administration, Rockville, MD. 20892; e-mail: [email protected]. Submitted October 1, 2001; accepted November 30, 2001. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby Supported by a grant from Japanese Foundation of AIDS Prevention (to Y.O.), marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734. and by a Leukemia and Lymphoma Society of America translational research grant 6540-00 and American Foundation for AIDS Research (AmFAR) grant © 2002 by The American Society of Hematology

BLOOD, 15 APRIL 2002 ⅐ VOLUME 99, NUMBER 8 2851 2852 OKAMATO et al BLOOD, 15 APRIL 2002 ⅐ VOLUME 99, NUMBER 8

Interleukin-7 (IL-7), which was originally reported as a pre-B selected and used for TOC. Each thymic fragment was cultured on cell growth factor,23 is produced by stromal cells in the thymus and polyethylene terephthalate track-etched trans-well membranes (Becton bone marrow and appears to play a role at multiple stages of T- and Dickinson, San Jose, CA) suspended in 12-well plates in UltraCULTURE B-lymphocyte development.24,25 In mice, the IL-7 receptor (IL-7R) fetal calf serum (FCS)–free medium (Biowittaker, Walkersville, MD) supplemented with 50 U/mL penicillin and 50 mg/mL streptomycin in the is first expressed in lymphoid lineage-restricted progenitors in bone presence or absence of recombinant human IL-7 (Peproteck, Rocky Hill, marrow26 and later can be detected in various tissues including NJ). The cultures were maintained in a 5% CO2 incubator at 37°C. thymus.27 IL-7R is composed of the IL-7R␣27 chain and the common cytokine receptor ␥ chain,28 the latter of which is the Treatment of NOD-SCID-hu mice with IL-7 indispensable subunit receptor for several lymphoid-related cytokines such as IL-2, IL-4, IL-9, and IL-15.29 Several studies have The NOD/LtSz-scid mice were purchased from Jackson Laboratory (Bar shown the essential role of this cytokine in the survival and normal Harbor, ME). Mice were implanted with pieces (ϳ2mm3 each) of human differentiation of thymocytes into mature naı¨ve T cells in mice. It fetal thymus and liver under the kidney capsule as previously described39,40 has been suggested that IL-7 is a cofactor for V(D)J rearrangement and maintained under specific pathogen-free conditions. Twenty-eight days after implantation, the size of the grafts was checked and the mice were of the TcRb gene locus30 and that it is also required for TcRb D-J 31 accordingly divided into 4 groups with similarly sized thymic grafts. Four rearrangement. In mice with targeted deletions of the IL-7 gene days later, 5 mice from each group were injected intraperitoneally twice Ϫ/Ϫ (IL-7 ), there was a 20-fold reduction in the number of pro–T daily with 100 ng each of IL-7 or phosphate-buffered saline. This was 32 cells, yet the percentage of TcR␣␤ thymocytes was relatively continued daily for 10 days. On days 7 and 21 after the last injection, normal,25,32 whereas the number of TcR␥␦ thymocytes was sub- IL-7–treated and control mice were autopsied and the graft, blood, spleen, stantially reduced.32 In mice with deletions of the IL-7R gene and mesenteric lymph nodes were recovered for evaluation of TREC levels (IL-7R␣Ϫ/Ϫ and ␥cϪ/Ϫ), the number of TcR␣␤ thymocytes was and phenotypic analysis. markedly decreased and TcR␥␦ thymocytes were completely absent.33,34 On the other hand, in humans, it has been reported that Flow cytometry mutations that prevent the expression of IL-7R␣ chain and ␥c result 35,36 Thymocytes were stained with combinations of the following directly in severe combined immunodeficiency (SCID). Thus, it appears labeled antihuman mouse monoclonal antibodies and isotype-matched that IL-7 is necessary for the survival and proliferation of early controls: CD1a (HI149), CD3 (SK6), CD4 (SK3), CD8 (SK1) (Becton thymic progenitor cells and production of TcR␥␦ T cells, although Dickinson); CDw127 (IL-7R␣) (R34.34) (Beckman Coulter, Brea, CA); it may not be unconditionally required for rearrangement of CD3 (UCHT1), Ki67 (B56), BrdU (3D4), annexin V, CD45RA (HI00), (BD TcRa and b genes in mice. However, the regulatory mechanism Pharmingen, San Diego, CA); and BCL-2 (124) (DAKO, Glostrup, of TCR␣␤ or ␥␦ rearrangement in human thymus and the roles Denmark). After staining, the cells were fixed in 1% paraformaldehyde and of IL-7 in thymopoiesis are as yet not fully understood. 4-color flow cytometry was performed using a FACSCalibur (Becton Importantly, it has been recently shown that IL-7 levels are Dickinson) flow cytometer. For Ki67 and BCL-2, the cells were first stained increased during T lymphopenia in HIV infection, strongly for surface CD3, CD4, and CD8 and then fixed and permeabilized using the suggesting that IL-7 may play a major role in both peripheral Cytofix/Cytoperm kit (BD Pharmingen) prior to staining of these intracellular proteins. Bromodeoxyuridine (BrdU) staining was carried out after and central T-cell homeostasis.37,38 staining for surface antigens using the BrdU Flow Kit (BD Pharmingen) We evaluated the effects of exogenous IL-7 on human thymopoi- according to the manufacturer’s instructions. The data were analyzed using esis in vitro using human thymic organ culture (TOC) and in vivo CellQuest software (Becton Dickinson). with NOD/LtSz-scid mice implanted with human thymus and liver (NOD-SCID-hu). The addition of IL-7 to TOC increased thymo- Sorting of thymocytes cyte proliferation and TREC levels and decreased apoptosis in thymuses obtained from fetuses and infants. In vivo, exogenous After mechanical disruption of thymus fragments, the cells were labeled IL-7 enhanced TREC levels in thymic grafts in NOD-SCID-hu with CD3 MicroBeads (Miltenyi Biotech, Auburn, CA) and separated into ϩ Ϫ mice. Our results indicate that IL-7 results in increased TCR CD3 and CD3 populations using separation columns (Miltenyi Biotech) according to the manufacturer’s instructions. Thymic subsets were sorted rearrangement in human thymus, both in vitro and in vivo, and by FACStar cell sorter (Becton Dickinson) after being stained for CD1a, suggest that immune reconstitution in humans could be augmented CD3, CD4, and CD8. through stimulating thymus-dependent T-cell generation with exogenous IL-7. Quantitative polymerase chain reaction of TRECs

The sorted cells were lysed in 100 ␮g/mL proteinase K (Roche Diagnostics, Indianapolis, IN) for 3 hours at 56°C and then 20 minutes at 95°C. TREC Materials and methods levels were measured by real-time quantitative polymerase chain reaction (PCR) using the 5Ј-nuclease (TaqMan) assay in an AB17700 system Thymus and liver (Perkin-Elmer, Norwalk, CT). As previously described,16 each 25-␮L Human fetal thymus and liver tissue at 18 to 22 weeks of gestation were reaction contained 5 ␮L cell lysate, and the ﬁnal concentration of each obtained from Advanced Bioscience Resources (Alameda, CA) and were component was as follows: 1.0 times reaction buffer, 20 U/mL platinum taq processed within 24 hours of harvest. Newborn, infant, and adult thymuses polymerase (Gibco BRL, Grand Island, NY), 3.5 mM MgCl2, 0.2 mM were obtained from patients undergoing corrective cardiac surgery or adult dNTPs, 500 nM each primer, 150 nM probe, and Blue-636 reference thymectomy (Children’s Medical Center or Parkland Memorial Hospital, (Megabases, Chicago, IL). The sense and antisense primers were 5Ј- Dallas, TX) and were processed within 6 hours of harvest. cacatccctttcaaccatgct-3Ј and 5Ј-cctaaaccctgcagctggc-3Ј, respectively, and the probe was FAM-acacctctggtttttgtaaaggtgcccact-TAMRA (MegaBases). TOC PCR conditions were 95°C for 5 minutes followed by 40 cycles of 95°C for 30 seconds and 60°C for 1 minute. A standard curve was plotted and TREC Thymus tissue was dissected into pieces of about 2 mm3 so that every piece values for samples were calculated using ABI7700 software. Samples were was likely to contain cortex and have similar cellularity. In adult samples, analyzed in duplicate. TRECs are present at 0, 1, or 2 copies per diploid cell only the lobes that, on visual inspection, contained cell-dense areas were and there is no pseudogene sequence. BLOOD, 15 APRIL 2002 ⅐ VOLUME 99, NUMBER 8 IL-7 AND HUMAN THYMUS 2853

Results

TREC levels in FACS-sorted thymocyte subsets in the fresh human thymus

To investigate the timing of TREC generation and dilution during thymic development in vivo, we measured TREC levels in sorted thymocyte subsets from a newborn infant. The cells were separated by magnetic cell sorting (MACS) and flow cytometry based on surface phenotype. Commitment of progenitor cells to the T-cell lineage occurs at or around the transition of CD1aϪCD34ϩ thymocytes into CD1aϩCD34ϩ thymocytes.41,42 Cells were sorted by flow cytometry for CD1aϩ/ϩϩCD3Ϫ and CD3ϩ subsets so as to exclude the CD1aϪCD3Ϫ subset, which contains precursors for natural killer (NK) cells and thymic dendritic cells.43-45 Quantita- tive PCR demonstrated that the TREC level in unfractionated thymocytes was 12 194/10 000 cells (Figure 1). No TRECs were detected at the CD3ϪCD4ϪCD8Ϫ triple negative (TN), CD3ϪCD4lowCD8Ϫ,orCD3ϪCD4ϩCD8Ϫ stages of thymocyte Figure 2. IL-7R␣ (CD127) expression in thymuses from subjects of varying ages. Freshly isolated thymocytes from a 22-week gestation fetus, a 2-month-old development. However, the highest level of TRECs was detected infant, and a 14-year-old youngster were analyzed by flow cytometry for expression within CD1aϩ cells that are CD3ϪCD4ϩCD8ϩ double positive of IL-7R␣ (CD127) on multiple cell types. (CD3Ϫ DP; 14 692/10 000 cells), confirming previous experiments that address the timing of TCRD gene rearrangement.46 This indicates that the excision leading to TRECs that commits a cell to vary depending on the possible TCR deletion on only one allele in the TCR␣␤ lineage occurs concomitant with the expansion of part of the cells.48 Because cellular divisions after TREC genera- CD3Ϫ DP thymocytes. If we assume that TCRD deletion occurs in tion will dilute TRECs, these results indicate that the initiation of both alleles,47 then a TREC level of 14 692/10 000 cells would cell expansion precedes TREC generation in CD3Ϫ DP cells. High indicate than an average of 75% of cells have undergone rearrange- levels of TREC were still detected in the CD3ϩ DP subset, which ment of both alleles to generate TREC. However, this value may had undergone expansion (6900/10 000 cells), and also in the more mature CD4ϩ and CD8ϩ single positive (SP) subsets (6064 and 4099/10 000 cells, respectively). The decrease in TRECs in mature cells is likely to represent dilution secondary to cellular proliferation that occurs during the processes of positive and negative selection. There was no evidence to suggest that TREC generation still occurs after the CD3ϩ stage.

IL-7R␣ expression in thymocyte subsets

As a basis for studying the role of IL-7 in TREC generation in human thymus, IL-7R␣ expression was examined by flow cytometry on thymocytes from a fetus (22 weeks’ gestation), a 2-month- old infant, and a 14-year-old youth (Figure 2). IL-7R␣ expression was highest in immature CD3ϪCD4lowCD8Ϫ cells (89.0%-95.5%), just before TREC generation (Figures 1 and 2). Because cells with this phenotype are thought to be initiating the process of TCR␣␤ or ␥␦ lineage commitment and are known to undergo proliferation,49 these results suggest the involvement of IL-7R␣ in regulating the proliferation of thymocytes and their commitment to TCR␣␤ or ␥␦ lineage. Although IL-7R␣ expression tended to decrease after the Figure 1. TREC levels in FACS-purified thymocyte subsets from the human Ϫ low Ϫ thymus. TREC levels were measured in FACS-sorted cells from a fresh thymus of a CD3 CD4 CD8 stage, it was still fairly high (52.2%-85.9%) in newborn infant. For CD3Ϫ cells, the thymocytes were first depleted of CD3ϩ cells by CD3Ϫ DP cells regardless of the age of the thymus. IL-7R␣ MACS and then sorted by FACS for the subsets based on CD4 and CD8 expression expression was lowest in CD3ϩ DP cells (31.4%-57.7%) and then (dot plot, upper right) to more precisely determine the timing of peak TREC ϩ production. Cells were also sorted for CD1aϩ/ϩϩ cells so as to exclude the increased again in CD3 SP cells (43.6%-69.2%). CD1aϪCD3Ϫ subset, which may contain precursors for NK cells and thymic dendritic cells. The sorted cells were subjected to TREC assay. The CD3ϩ-depleted fraction The effects of IL-7 on proliferation and apoptosis (shown as R4 in the histogram, upper left) contained cells expressing CD3 at the of thymocytes intermediate level (referred to as CD3low) as well as CD3Ϫ cells. On the other hand, Ϫ Ϫ/low for measuring the frequency of each subpopulation in CD3 and CD3 popula- Several studies have suggested that IL-7 is a physiologic survival tions, we analyzed whole thymocytes from the same thymus sample using gate R1. 24,50-52 For CD3ϩ subsets, the whole thymocytes were analyzed for frequency measurement factor for early lymphoid progenitor cells. The evaluation of using gates R2 or R3 and then sorted for the TREC assay. Therefore, it should be TREC levels in each thymic subpopulation will be affected by noted that the actual frequency of CD3Ϫ/low CD4ϩCD8ϩ and CD3ϩCD4ϩCD8ϩ cells proliferation and apoptosis of thymocytes. To evaluate the in vitro should be slightly higher and lower, respectively, than the values shown here (36.4% and 35.7%, respectively). The TREC values are expressed as copies per 10 000 effects of exogenous IL-7 on TREC generation in our TOC system, cells. These data represent 3 different experiments. we first assessed the effect of IL-7 on thymocyte proliferation by 2854 OKAMATO et al BLOOD, 15 APRIL 2002 ⅐ VOLUME 99, NUMBER 8

respectively) but also in more mature CD3ϩCD4ϩ SP (6.6 Ϯ 3.3- fold) and CD3ϩCD8ϩ SP cells (18.8 Ϯ 8.6-fold). This effect was dependent on IL-7 dose (data not shown). We next assessed the effects of IL-7 on apoptosis in TOCs by measuring expression of annexin V after 4 days of TOC in the presence or absence of IL-7. IL-7 signiﬁcantly lowered annexin V expression by about 50% in CD3ϪCD8Ϫ immature thymocytes (Figure 3B, upper and lower panels), which were pre–TREC- generating cells (Figure 1). We then examined BCL-2, a well- established protein, which is involved in the suppression of apoptosis. It has been reported that BCL-2 expression during T-cell differentiation is multiphasic; it is high in CD3Ϫ immature thymocytes, down-regulated in CD4ϩCD8ϩ thymocytes to facili- tate selection process, and up-regulated in mature CD3ϩ SP thymocytes thus enhancing prolonged survival.53 As expected, IL-7 enhanced BCL-2 expression in CD3Ϫ immature thymocytes in a dose-dependent way (Figure 3C). These results were consistent with previous studies in mice demonstrating that short-term culture with IL-7 of immature thymocytes from IL-7Ϫ/Ϫ mice caused up-regulation of BCL-2 and increased cell survival.51 Thus, in human thymus, IL-7 led to an increase in proliferation and decrease in apoptosis of immature CD3Ϫ to CD3low cells, which correlated with the increased expression of BCL-2.

The in vitro effects of IL-7 on TREC generation in TOCs

The effects of IL-7 on TREC generation were examined in TOCs. After culturing newborn thymus in the presence (10 and 50 ng/mL) or absence of IL-7 for 4 days, thymocytes were separated into CD3Ϫ and CD3ϩ fractions by MACS (Figure 4A). The majority of CD3low cells were contained in the CD3Ϫ fraction (Figure 4A), whereas most of CD3ϩ DP cells were collected into the CD3ϩ fraction (data not shown). Figure 4B shows the TREC frequency in

Figure 3. Proliferation and apoptosis of thymocytes in response to IL-7. (A) The incorporation of BrdU was measured in thymocytes in the presence (10 ng/mL) or absence of IL-7 on day 4 of TOC. Upper panel shows representative results from 5 different experiments. Ratio of IL-7 to control is shown in the lower panel. (B) The inhibition effect of IL-7 on apoptosis of thymocytes was evaluated by ﬂow cytometry by the expression of annexin V after 4 days of TOC. Upper panel shows the representative result from 5 different experiments. Lower panel shows the percent inhibition of annexin V expression by IL-7 to control TOC. BCL-2 expression was evaluated in TOC with 1, 10, and 50 ng/mL IL-7. Data are expressed as mean value Ϯ SD in triplicate. measuring the incorporation of BrdU. The upper panel of Figure 3A represents several experiments using different thymuses. The Figure 4. The in vitro effects of IL-7 on TREC generation in TOCs. Thymocytes rate of BrdU incorporation was highest in CD3Ϫ/lowCD4ϩCD8Ϫ from a newborn thymus were separated by MACS for CD3Ϫ and CD3ϩ subsets cells and low in mature CD3ϩ cells (Figure 3A). We observed a before or after 4 days of TOC in the absence or presence of IL-7 (10 and 50 ng/mL) and TREC levels were measured. (A) The representative data of CD3 expression is marked increase in BrdU uptake in most thymocyte populations shown in CD3Ϫ (thin line) and CD3ϩ (bold line) fractions sorted by selection column with the administration of 10 ng/mL IL-7 (Figure 3A). Increased from preculture and day 4 of TOC in the absence or presence of IL-7 (10 ng/mL). (B) BrdU uptake with IL-7 was observed consistently in 5 different TREC levels in the 2 fractions were measured. Three separate experiments were performed. Each experiment was performed in quadruplicate with all 3 concentra- TOCs (Figure 3A, lower panel), not only in immature cells tions of IL-7. A representative result is shown with error bars pertaining to the (4.0 Ϯ 3.3-fold and 2.3 Ϯ 0.8-fold in TN and CD3ϪCD4lowCD8Ϫ, experiment. BLOOD, 15 APRIL 2002 ⅐ VOLUME 99, NUMBER 8 IL-7 AND HUMAN THYMUS 2855

TOCs. IL-7 increased TREC levels in both CD3Ϫ and CD3ϩ checking the size of the grafts. TREC levels in thymocytes from the subsets in a dose-dependent way. Although there was a difference grafts were significantly higher in the IL-7–treated group (14 276 Ϯ between 10 ng/mL IL-7–treated and untreated TOC, the only 1290 and 14 344 Ϯ 2449 for day 7 and day 21, respectively) than statistically significant difference existed between the 50 ng/mL the control group (8135 Ϯ 1070 and 9877 Ϯ 1869 for day 7 and day and untreated TOCs (P Ͻ .09 and P Ͻ .001 in CD3Ϫ and CD3ϩ 21, respectively) at day 7 (P Ͻ .002) and day 21 (P Ͻ .007) after subsets, respectively). the last administration of IL-7 (Figure 5B). Although the average If IL-7 were to be used as a therapeutic agent to improve graft weight (Figure 5C) was higher in IL-7–treated mice after 7 immune reconstitution, its effect would have to be maintained in days, the difference between the 2 groups was not statistically postnatal thymuses. The effects of IL-7 on TREC levels were significant either after 7 or 21 days. Moreover, there was no change further evaluated in TOCs using thymuses from subjects of by IL-7 treatment in the percentage of Ki67ϩ cells in any of the differing ages. Fetal (18, 19, and 22 weeks’ gestation), newborn (2 subsets except for CD3ϩ DP and CD3ϩ CD8 SP at day 7 (Figure and 15 day, and 3, 4, 7, and 11 month), and infant (3 and 5.6 year) 5D). Also, there was no significant increase either in the percentage thymuses were placed in TOCs in the presence of IL-7, and TRECs of human CD3ϩ cells or TREC levels in the peripheral blood after were measured (Table 1). In all TOCs, TREC levels increased in the IL-7 treatment (data not shown). presence of IL-7 (1.3 Ϯ 0.1-fold for whole cells, 2.0 Ϯ 0.8-fold for CD3Ϫ fractions, and 1.5 Ϯ 0.2-fold for CD3ϩ fractions).

The in vivo effects of IL-7 on TREC generation Discussion in NOD-SCID-hu mice It is well established that IL-7 exerts potent effects on T-cell Several studies have demonstrated the in vivo effects of IL-7 on progenitors and is required for T-cell development. It is critical for mouse thymopoiesis.54,55 In the present study we used NOD- homeostatic proliferation and survival of not only thymocytes but SCID-hu chimeric mice to measure the in vivo effects of IL-7 on also naı¨ve T cells in the peripheral blood and lymph nodes.56 In the human thymopoiesis. Twenty-eight days after engraftment of present study we used the quantitative TREC assay to assess more human fetal thymus and liver under the mouse kidney capsule, the deﬁnitively the effects of exogenous IL-7 on TCR rearrangement in growth of thymic grafts was readily apparent; typically, a 2-mm3 thymocytes using both in vitro TOCs and thymic engraftments in fragment grew up to a diameter of 5 to 7 mm. These grafts NOD-SCID mice. contained comparable levels of TRECs per thymocytes to those of To understand the timing of TREC generation and its dilution native human thymuses (data not shown), indicating the generation during thymic development, we quantiﬁed TREC level in each of new thymocytes in the grafts. More importantly, we were able to thymic subpopulation. We found the highest level of TREC in the detect human CD45ϩ cells in the peripheral blood and spleen. Most CD1aϩCD3Ϫ DP stage. Recently, TCR␤ selection in the human of these human cells were positive for CD3 (data not shown) and thymus was reported to be initiated at the transition of represented 1.0% and 1.3% of the total lymphocytes in PBMCs and CD3ϪCD4ϩCD8Ϫ into the CD4ϩCD8␣ϩ␤Ϫ stage,57 whereas TCRA spleen, respectively (Figure 5A, upper panel). These CD3ϩ cells gene expression has been known to be restricted to CD3ϩ DP and comprised mature CD4ϩ and CD8ϩ SP cells (Figure 5A, middle later stages of T-cell development. Together with these, our data panel), the majority of which were CD45RAϩ, suggesting that they suggest that TCRB gene rearrangement precedes and induces the were naı¨ve T cells (Figure 5A, bottom panel). The frequency of initiation of TREC generation, followed by TCRA rearrangement. human CD3ϩCD45RAϩCD4ϩ or CD3ϩCD45RAϩCD8ϩ T cells TREC generation may represent a decisive step in the ␣␤ versus ␥␦ correlated positively with the size of the graft (data not shown). lineage commitment of differentiating thymocytes because this is We treated NOD-SCID-hu mice with 100 ng IL-7 (n ϭ 11) or an intermediate rearrangement between TCRD and TCRA gene saline (n ϭ 8) twice daily for 10 days beginning 4 days after rearrangement, which excises most of the TCRD locus.58 The

Table 1. The effect of IL-7 on TREC levels in TOCs using thymuses of differing ages TREC/10,000 cells Day 4 or Day 5* Whole CD3Ϫ CD3ϩ TOC no. Age of thymus Control IL-7 Ratio† Control IL-7 Ratio† Control IL-7 Ratio†

1 Fetal 18 wk-1 4 738 6 074 1.3 4 120 13 561 3.3 ND ND ND 2 18 wk-2 ND ND ND 4 047 11 318 2.8 ND ND ND 3 19 wk ND ND ND 3 970 4 883 1.2 6 618 8 347 1.3 4 22 wk ND ND ND 1 703 3 885 2.3 3 310 5 321 1.6 5 Newborn 2 d 10 639 15 265 1.4 7 733 13 462 1.7 7 808 12 697 1.6 6 15 d 9 790 12 333 1.3 2 080 5 044 2.4 7 507 9 751 1.3 7 3 mo 4 682 6 943 1.5 ND ND ND ND ND ND 8 4 mo 5 175 6 756 1.3 2 616 3 232 1.2 ND ND ND 9 7 mo 6 071 7 503 1.2 2 389 5 585 2.3 9 700 14 979 1.5 10 11 mo 5 820 7 884 1.4 4 195 4 387 1.0 5 768 8 416 1.5 11 Infant 3 y 8 590 12 183 1.4 ND ND ND ND ND ND 12 5.6 y 8 297 10 200 1.2 ND ND ND ND ND ND Mean Ϯ SD 1.3 Ϯ 0.1 2.0 Ϯ 0.8 1.5 Ϯ 0.2

ND indicates not done. *TOC no. 3 and 11 were cultured for 5 days and others were cultured for 4 days. †IL-7(ϩ)/IL-7(Ϫ). 2856 OKAMATO et al BLOOD, 15 APRIL 2002 ⅐ VOLUME 99, NUMBER 8

Figure 5. The in vivo effects of IL-7 on TREC generation in NOD-SCID-hu mice. Four weeks after implantation of human fetal thymus and liver, NOD-SCID-hu mice were treated with 100 ng IL-7 twice daily for 10 days. Seven (control, n ϭ 3; IL-7, n ϭ 4) and 21 days (control, n ϭ 5; IL-7, n ϭ 7) after the last injection, the grafts, spleen, and PBMCs were recovered from the mice for phenotypic analysis and TRECs. The upper panels of panel A indicate percentage of human CD3ϩ cells and the panels in the middle row of panel A indicate human CD4ϩ and CD8ϩ expression on the CD3ϩ population of PBMC (left) and spleen (right). The bottom ﬁgures of panel A show CD45RAϩ cells on CD3ϩCD4ϩ or CD3ϩCD8ϩ populations of PBMCs (left) and spleen (right). The TREC levels of grafts and the graft weights in control and IL-7–treated mice are shown in panels B and C, respectively. The percentages of Ki67ϩ cells in each subpopulation of grafts are shown in panel D.

only apparent reason for this rearrangement is to delete the expression in any of the subsets did not show a significant change TCRD locus to prepare the allele for subsequent TCRA gene by IL-7 treatment (Figure 5C and 5D, respectively). Considering rearrangement. Thus, our finding that IL-7 increases TREC that IL-7 can exhibit distinct effects on T cells at different levels of thymocytes most likely indicates that IL-7 eventually concentrations,60 one possible interpretation of our data would be leads to the enhancement of induction of TCRA and TCRB gene that the endogenous human IL-7 in these grafts was sufficient to rearrangement. maintain the normal development of thymocytes, whereas the Although IL-7 increased TREC in our TOC system, we cannot addition of exogenous IL-7 further promoted TCR gene rearrange- definitively prove that this is due to the promotion of TREC ment and therefore TREC generation, but was not enough to generation in the individual thymocytes because TREC levels are promote further proliferation of immature cells. In this experiment also affected by the ability of IL-7 to regulate apoptosis and we did not measure the rate of apoptosis in the thymic grafts. proliferation of cells at the pre–TREC-generating stage.49,50,51,59 However, it is unlikely that this increase in TREC by IL-7 was Indeed, BrdU, annexin V, and BCL-2 staining in our TOC system caused solely by lengthening the survival of immature cells clearly demonstrated that exogenous IL-7 can enhance the prolifera- because the percentage of annexin Vϩ cells in fresh human thymus tion and prevent apoptosis of immature CD3Ϫ/low subsets (Figure 3) is typically as low as 0.5% to 4.0% in each subpopulation (Yukari which, according to our results (Figure 1), precede TREC genera- Okamoto unpublished data, May 1999). Therefore, the logical tion. It is quite possible, therefore, that IL-7 indirectly increases the interpretation is that IL-7 can directly stimulate TREC generation. TREC frequency in the thymus by promoting the proliferation and Based on transgenic mouse studies, it has been suggested that there survival of thymocytes pre-TREC generation. is a regulatory sequence near the human ␦Rec gene segment, which On the other hand, our in vivo experiments suggested another is recognized by putative regulatory proteins that induce ␦Rec pathway of IL-7 effects. Exogenous IL-7 was administered to rearrangement.61 Moreover, transcription of the T-early ␣ (TEA) NOD-SCID-hu mice, where the growth of human thymic grafts element is assumed to be responsible for opening the TCRAJ locus appeared to be fairly active. At 7 and 21 days after the last to the V(D)J recombinase complex.62,63 Further identification of administration, TREC levels in thymocytes from the grafts were regulatory mechanism of the ␦Rec-␺J␣ recombination event will significantly higher in the IL-7–treated group than the control eventually allow a determination of whether IL-7 has a direct effect group (Figure 5B). However, the graft weights and also Ki67 on intracellular processes that lead to enhanced TREC production. BLOOD, 15 APRIL 2002 ⅐ VOLUME 99, NUMBER 8 IL-7 AND HUMAN THYMUS 2857

In our NOD-SCID-hu mouse system, we observed no signiﬁ- practice it may be necessary to antagonize the effects of thymic cant increase for the percentage of human CD3ϩ cells in the atrophic factors.70 The decrease in thymocyte apoptosis mediated peripheral blood as a result of IL-7 treatment. It has been reported by high concentrations of exogenous IL-7 in CD3ϩ DP population that mouse NK cells affect the efﬁciency of engraftment of human may affect negative selection. Furthermore, it should also be cells in this model.64 Although NOD/LtSz-scid mice have rela- considered that high concentrations of exogenous IL-7 may break tively low NK cell activity,65 it is possible that IL-7 caused an unresponsiveness of residual autoreactive T cells in healthy individu- enhancement of this.66,67 Be that as it may, our data clearly show als by stimulating the anergic T cells that suppress the autoreactive that IL-7 increased TCR gene rearrangement and TREC genera- cells71 and may cause autoimmune disease. Nevertheless, our tion, which would suggest that thymopoietic function was aug- results provide a basis for understanding some of the regulatory mented in vivo. mechanisms of thymocyte development in the human thymus and Complete recovery of broad T-cell immunity after insults such encourage the further evaluation of cytokine-based immune recon- as HIV infection or chemotherapy will require the generation of stitution strategies, through the stimulation of thymus-dependent new naı¨ve T cells from the thymus.2,16,17,21 The administration of T-cell generation. thymopoietic cytokines may be able to improve immune reconstitution by augmenting thymic function in these situations. IL-7 is a major factor for thymopoiesis, and systemic administration of recombinant IL-7 to murine bone marrow transplant recipients has Acknowledgments been shown to normalize thymopoiesis and improve immune function after bone marrow transplantation.21,68,69 Furthermore, We thank Brenna Hill, Joseph Beckham, and Angie Mobley for endogenous IL-7 may play a major role in the homeostatic response technical help; Dr Michael Betts and Dr Takeshi Kurata for helpful to T lymphopenia.37 Our data suggest that IL-7 can enhance the advice; and Dr Steven Leonard for procurement of postnatal function of fetal and infant thymuses, although in therapeutic thymuses. References

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